The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/− 3000 and 46,000 +/− 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/− 3000, 46,000 +/− 3000, 25,000 +/− 7000 and 12,000 +/− 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/− 3000 Mr proteins and 17% less incorporation into 12,000 +/− 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/− 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS)

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